A major goal of biomedical basic research is to understand the developmental genetic basis of phenotypic variation. Investigations into the genetic basis of repeated phenotypic evolution in emerging model genetic taxa provide an opportunity to investigate whether, and how often, evolution proceeds through similar genetic and developmental changes and whether there are developmental genetic constraints on phenotypic evolution. The proposed research develops a research program designed to investigate the genetic and developmental basis of repeated shifts in floral pollination syndrome, a complex adaptive trait, in the plant genus Penstemon. These initial experiments will provide insights and valuable resources for the development of this research program. In this genus, there have been at least 15 independent evolutionary shifts from the ancestral bee pollination syndrome to a highly stereotypical hummingbird pollination syndrome. The first aim of the proposed research will develop genomic resources and map pollination syndrome divergence for a pair of closely related Penstemon species that differ in their pollination syndrome. Specifically, an F2 mapping population derived from a cross between these two species will be phenotyped for floral traits and genotyped using Multiplexed Shotgun Genotyping, a form of genotyping-by-sequencing. This will enable the construction of a linkage map and identification of quantitative trait loci underlying individual components of pollination syndrome. Genomic DNA from each focal species will be sequenced, assembled, and aligned to the linkage map to yield a set of ordered genome contigs, comprising a valuable resource for future experiments. The second research aim is to characterize the developmental basis of floral morphological divergence between three separate origins of the hummingbird syndrome by comparing cell number, size, and shape in floral organs across developmental stages for each hummingbird syndrome species relative to a related bee syndrome species. Methods for this aim include microscopy and in situ hybridization to assess patterns of histone4 gene expression, which marks active cell division. The final aim is to develop protocols facilitating stable genetic transformation in Penstemon, which will be a valuable tool for the functional verification of candidate genes in future experiments.